WO2020124831A1 - 一种构建高效枯草芽孢杆菌启动子的方法 - Google Patents

一种构建高效枯草芽孢杆菌启动子的方法 Download PDF

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WO2020124831A1
WO2020124831A1 PCT/CN2019/078816 CN2019078816W WO2020124831A1 WO 2020124831 A1 WO2020124831 A1 WO 2020124831A1 CN 2019078816 W CN2019078816 W CN 2019078816W WO 2020124831 A1 WO2020124831 A1 WO 2020124831A1
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promoter
seq
promoters
bacillus subtilis
tandem
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周哲敏
崔文璟
韩来闯
刘中美
周丽
郭军玲
燕宇
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Jiangnan University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/10—Vectors comprising a special translation-regulating system regulates levels of translation
    • C12N2840/105—Vectors comprising a special translation-regulating system regulates levels of translation enhancing translation

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  • the invention relates to a method for constructing an efficient Bacillus subtilis promoter, which belongs to the technical field of genetic engineering.
  • Bacillus subtilis is a Gram-positive model strain widely used for the expression of foreign proteins. It is widely used in the production of industrial enzyme preparations because it can efficiently express foreign proteins.
  • Bacillus subtilis promoters especially natural endogenous Bacillus subtilis promoters (such as the P43 promoter) have low activity and poor stability in expressing foreign genes. This defect severely restricts Bacillus subtilis The application of Bacillus in the field of efficient expression of foreign proteins.
  • people have used the idea of directed evolution to further screen and modify natural promoters, on the other hand, they have used genetic engineering methods and synthetic biology ideas to construct artificial synthetic promoters.
  • the modification of the natural promoter can greatly improve the promoter activity, there are still no rules to avoid some of the natural promoter's own defects, such as unstable expression and poor compatibility with other expression control elements. Therefore, the construction of efficient and stable artificial promoters has great application prospects for breaking through the bottleneck of natural promoter activity and improving the stability and compatibility of promoter elements.
  • the former relies on a highly active natural promoter, which also requires people to have a deeper understanding of the working principle of the promoter, and for the tandem promoter, if the new promoter contains multiple repeated Sequence fragments will adversely affect the stability of the promoter and expression vector; although the latter does not require an in-depth understanding of the working mechanism of the promoter, screening from a completely random sequence to the target promoter requires expensive high-throughput screening Instrumentation equipment, low screening efficiency. Therefore, providing a method for constructing efficient and stable promoters is of great significance for the efficient expression of foreign proteins.
  • a first object of the present invention is to provide a gene expression regulatory elements, including promoter and an artificial RBS series downstream of the tandem promoter is an artificial promoter P rpoB, P spoVG and at least two P sigW one in series, the nucleotide sequence of the promoter P rpoB, P spoVG and P sigW respectively as SEQ ID NO.1, SEQ ID NO.7 and shown in SEQ ID NO.11.
  • nucleotide sequence of the artificial tandem promoter is shown in any one of SEQ ID NO.17-SEQ ID NO.27
  • nucleotide sequence of the artificial tandem promoter is shown in any one of SEQ ID NO.29-SEQ ID NO.59.
  • the core region and the P rpoB P spoVG promoter when between the core region and the P rpoB P spoVG promoter are inserted into the spacer sequence 60bp, 75bp respectively obtained as SEQ ID NO.32, SEQ ID NO.33 the The new promoters shown are PAW-D60 and PAH-D75 .
  • the nucleotide sequence of the RBS is shown in any one of SEQ ID NO. 60-72.
  • the expression host of the gene of interest includes Bacillus subtilis.
  • the expression host of the target gene includes Bacillus subtilis 168, Bacillus subtilis WB400, Bacillus subtilis WB600, or Bacillus subtilis WB800.
  • the target gene includes a foreign gene or an endogenous gene.
  • the target gene includes an enzyme gene or a non-enzyme gene.
  • the second object of the present invention is to provide a carrier containing the above elements.
  • the third object of the present invention is to provide genetically engineered bacteria expressing the above vector.
  • the fourth object of the present invention is to provide a method for regulating the expression of a target gene in Bacillus subtilis, co-expressing the above-mentioned regulatory element with the target gene.
  • the protein of interest includes an enzyme.
  • the Bacillus subtilis includes Bacillus subtilis 168, Bacillus subtilis WB400, Bacillus subtilis WB600, or Bacillus subtilis WB800.
  • the fifth object of the present invention is to provide the application of the above regulatory elements or genetically engineered bacteria in the preparation of a protein of interest.
  • the sixth object of the present invention is to provide the application of the above regulatory elements or genetically engineered bacteria in the fields of food, pharmaceutical or chemical industry.
  • the present invention first screens and characterizes different sigma subunit recognition promoters, and selects among them the sigA, sigH, and sigW subunits that have the highest recognition activity (the recombinant plasmids containing different regulatory elements are transformed into Bacillus subtilis , Using the fluorescence intensity of the culture solution after the cultivation of recombinant bacteria to characterize the expression level of the target gene and the activity of the regulatory element), the promoter was obtained through the double tandem and triple tandem of the core region, the optimization of the core region spacer sequence, and the RBS redesign. Regulatory element with further enhanced activity.
  • the fluorescence intensity of P AW-D60 and P AH-D75 is 0.94 times and 1.03 times higher than that of P AW (fluorescence intensity is 20262a.u/OD 600 ) before modification; P WAH-D45 and P WAH-D75 fluorescence intensity (18245a .u./OD 600 ) is 0.87 times and 0.96 times higher than P WAH (fluorescence intensity is 10261a.u/OD600) before modification; P AWH-D30 and P WAH-D90 fluorescence intensity is higher than P AWH before modification ( Fluorescence intensity was 16879a.u/OD600) increased by 0.78 times and 0.78 times respectively.
  • the fluorescence intensity can reach 76216a.u/OD 600 , which is 0.85 times higher than P AH-D75 ; P WAH-D75 and RBS13 (SEQ ID NO. 70) 72) in combination, the fluorescence intensity of up 77751a.u / OD 600, increased 1.17 times higher than P WAH-D75; when the P AWH-D30 and RBS13 (SEQ ID NO.72) in combination, the fluorescence intensity of up 73781a.u /OD 600 , 1.45 times higher than PAWH-D30 .
  • the method provided by the present invention, we can use a simple promoter design and modification method to obtain a promoter with higher activity, stronger design and compatibility.
  • the method is simple and easy to implement, and has broad application prospects in the construction of foreign protein high-efficiency expression systems and synthetic biology research.
  • Figure 1 Screening and characterization of the core region of a natural endogenous promoter recognized by different sigma subunits.
  • FIG. 1 Tandem promoter construction and activity characterization.
  • Figure 3 Spacer sequence optimization of tandem promoter core region a: Schematic diagram of spacer sequence optimization; b: P AH spacer sequence optimization; c: P WAH spacer sequence optimization; d: P AWH spacer sequence optimization.
  • Figure 4 a promoter and RBS compatibility detection, a: P rpoB promoter RBS design; b: P spoVG promoter RBS design; c: P sigW promoter RBS design; d: P AH-D75 promoter, RBS design; E : P WAH-D75 promoter RBS design; f: P AWH-D30 promoter RBS design.
  • Promoter cloning method design a primer containing a promoter sequence, using the E. coli-Bacillus subtilis shuttle vector pB-sfGFP (ie pBSG03, with the sfGFP reporter gene (Genbank ID: AVR55189.1), see Guan C ,Cui W,Cheng J,et al.Construction and development of auto-regulatory gene expression system in Bacillus subtilis[J].Microbial Cell Factories,2015,14(1):150) as a template, PrimeSTAR MAX DNA polymerase ( (Purchased from Takara, article number: R045Q) Perform whole plasmid PCR, PCR program: pre-denaturation 98 °C 1min, cycle of denaturation 98 °C 30s, annealing 50 °C 30s, extension 72 °C 1min, a total of 30 cycles, the last 72 °C extension 10min . Afterwards, the plasmid template was removed by digestion with restriction enzyme Dp
  • the detection method of sfGFP fluorescence intensity the sample is centrifuged at 12000 ⁇ g for 2 min, the bacterial cells are collected, washed 3 times with PBS buffer, diluted with PBS to a certain concentration of bacterial cell suspension, 200 ⁇ L is taken to a 96-well microtiter plate and placed SynergyTM H4 fluorescence microplate reader detects fluorescence. Excitation light 485nm, absorption light 528nm, fluorescence detection.
  • Medium LB medium (gL -1 ): Tryptone 10, NaCl 10, yeast extract 5, pH 7.0, and add agar powder 20 when preparing the solid medium.
  • Transformation method of Bacillus subtilis 168 single-inoculated Bacillus subtilis 168 inoculated into 2mL SPI medium and cultured in a shaker at 37°C for 12-14h; take 100 ⁇ L from the culture and inoculated into 5mL SPI medium, 37 OD 600 was measured after 4-5h incubation on a shaker at °C.
  • Example 1 Cloning and characterization of single promoters recognized by different sigma subunits
  • the cloned promoter core region includes the -10 region, -35 region of the above promoter and a transcription initiation site (TSS) totaling 70 bp.
  • the promoter sequence to be screened and identified is designed on the primers (see Table 2), the promoter sequence is introduced into the vector backbone pB-sfGFP by the method of whole plasmid PCR, and then the cloned is constructed by DpnI digestion, purification and assembly, transformation With a single promoter sfGFP expression plasmid.
  • the plasmid expressing the single promoter was transformed into Bacillus subtilis strain 168, and the obtained recombinant Bacillus subtilis was cultured in LB medium at 37°C and 200 rpm for 24h to detect the expression level of sfGFP, and the start was judged by the level of the fluorescent signal of sfGFP The level of child activity.
  • the results are shown in Table 1 and shown in SigA promoter identified, P rpoB promoter highest activity, identified the SigH promoter, P spoVG highest activity 3; SigW identified promoter, P sigW highest activity (FIG. 1).
  • the P rpoB, P spoVG P sigW in series and designed with Table 5 primers embodiment three plasmids constructed in Example 1 with promoter P rpoB, P spoVG P sigW as templates and do the whole plasmid containing the PCR construct
  • the tandem promoter is named according to the type and sequence of the tandem core region to obtain the double tandem promoters P AH , PAW , PHA , PHW , PAW , PWA and PWH (nuclear The nucleotide sequences SEQ ID NO.17-SEQ ID), three tandem promoter P AHW, P AWH, P HAW , P HWA, P WAH and P WHA shown NO.22 (nucleotide sequences shown in SEQ ID NO.23-SEQ ID NO.28), see Table 5 for the construction of primers.
  • the constructed recombinant plasmid was transformed into Bacillus subtilis 168.
  • the obtained recombinant Bacillus subtilis was cultured in LB medium at 37°C and 200 rpm.
  • the fluorescence signal was detected after 6h, 12h and 24h, and the promoter was judged by the level of the fluorescence signal of sfGFP The level of activity.
  • Table 6 The total fluorescence intensity after cultivation of recombinant bacteria with tandem promoter and tandem promoter sfGFP expression plasmids
  • the fluorescence intensities of recombinant bacteria with sfGFP expression plasmids containing double-tandem and triple-tandem promoters after 6, 12, and 24 hours of culture are shown in Figure 2 and Table 6.
  • the activities of double-tandem promoters and triple-tandem promoters are more than single promoters. All of them showed different degrees of improvement, and most of the activity of the triple tandem promoter was higher than that of the double tandem promoter, and the transformation of the P WHA promoter plasmid into Bacillus subtilis was unsuccessful.
  • the P AH with higher activity in the double tandem promoter and the P WAH and PAHH with higher activity in the triple tandem promoter were selected as the next step of transformation materials.
  • Insert spacer sequences of different lengths between the core regions of the promoter ( Figure 3a), and set the spacer sequence length to 15bp, 30bp, 45b, 60bp, 75bp and 90bp to obtain AH-D15, AH-D30, AH-D45, AH-D60, AH-D75, AH-D90, WAH-U15, WAH-U30, WAH-U45, WAH-U60, WAH-U75, WAH-U90, WAH-D15, WAH-D30, WAH-D45, WAH- D60, WAH-D75, WAH-D90, AWH-U15, AWH-U30, AWH-U45, AWH-U60, AWH-U75, AWH-U90, AWH-D15, AWH-D30, AWH-D45, AWH-D60, AWH-U75, AWH-U90, AWH-D15, AWH-D30, AWH-D45, AWH
  • the promoter sequences shown in SEQ ID NO.29-SEQ ID NO.59 were cloned on the pB-sfGFP vector, and then the recombinant plasmid was transformed into Bacillus subtilis 168 for detection, and the obtained recombinant Bacillus subtilis was in LB medium After incubation at 37°C and 200 rpm for 24 hours, the fluorescence signal was detected, and the level of promoter activity was judged by the level of the fluorescence signal of sfGFP.
  • Examples 1 and promoters in Example 3 P rpoB, PspoVG, P sigW, P AH-D75, P WAH-D75 and P AWH-D30 and 13 are combined RBS.
  • the RBS1-13 sequence (nucleotide sequences are shown in SEQ ID NO.60-SEQ ID NO.72 respectively) were cloned on a vector containing a tandem promoter, and then the recombinant plasmid was transformed into Bacillus subtilis 168, obtained Recombinant Bacillus subtilis was cultured in LB medium at 37°C and 200 rpm for 24 hours to detect the fluorescent signal, and the level of sfGFP fluorescent signal was used to judge the level of the activity of the regulatory element.
  • the fluorescence intensity can reach 76216a.u/OD 600 , which is 0.85 times higher than P AH-D75 ; P WAH-D75 and when RBS13 (SEQ ID NO.72) in combination, the fluorescence intensity of up 77751a.u / OD 600, increased 1.17 times higher than P WAH-D75; when the P AWH-D30 and RBS13 (SEQ ID NO.72) combinations, fluorescent The intensity can reach 73781a.u/OD 600 , which is 1.45 times higher than PAWH-D30 . It shows that the design of tandem promoter RBS can also enhance the expression of target genes.

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Abstract

提供了一种人工构建枯草芽孢杆菌启动子的方法以及构建获得的基因表达调控元件。该方法将不同sigma亚基识别的天然启动子串联得到一些双串联、三串联启动子，并在启动子串联的基础之上优化了启动子核心区之间的间隔序列长度进一步提升了启动子的活性，最后将启动子进行不同的RBS设计。该方法可以提升启动子和其他基因表达调控元件之间的兼容性，也对外源基因的表达进行了可控的调节。

Description

一种构建高效枯草芽孢杆菌启动子的方法 技术领域
本发明涉及一种构建高效枯草芽孢杆菌启动子的方法，属于基因工程技术领域。
背景技术
枯草芽孢杆菌是广泛应用于表达外源蛋白的革兰氏阳性模式菌株，其因能够高效表达外源蛋白在工业酶制剂生产方面有广泛应用。但是，目前应用的枯草芽孢杆菌启动子，尤其是天然的内源枯草芽孢杆菌启动子(如P43启动子)活性较低、表达外源基因的稳定性较差，这一缺陷严重制约了枯草芽孢杆菌在高效表达外源蛋白领域的应用。为了克服这一缺陷，近些年人们一方面利用定向进化的思路对天然启动子进一步筛选和改造，另一方面利用基因工程方法和合成生物学思路构建人工合成启动子。其中改造天然启动子虽然能大幅度提升启动子活性，但仍无法规避天然启动子自身的一些缺陷，如表达不稳定、与其他表达调控元件兼容性不强等。因此，构建高效稳定的人工启动子对于突破天然启动子活性瓶颈、提升启动子元件稳定性和兼容性方面有巨大的应用前景。
目前，构建人工启动子主要有两种策略，一种是利用天然启动子为基本骨架，筛选高活性天然启动子并进行精简、改造、重排、组合，进而构建出新的包含天然启动子骨架的高效人工启动子。另外一种策略是全人工合成随机的一定长度的DNA序列，克隆到启动子筛选载体上，借助高通量筛选设备和高通量筛选方法，筛选出众多随机序列中具有启动子功能的全人工合成序列作为启动子元件使用。这两种策略各有明显劣势，前者依赖高活性的天然启动子，也需要人们对启动子的工作原理有较为深入的认识，并且对于串联启动子来讲，新启动子如果包含多个重复的序列片段，将会对启动子和表达载体的稳定性带来不利影响；后者虽然不需要深入理解启动子的工作机制，但是从全随机序列中筛选到目标启动子需要昂贵的高通量筛选仪器设备，筛选效率低。因此，提供一种构建高效稳定启动子的方法，对于高效表达外源蛋白具有重要意义。
发明内容
本发明的第一个目的是提供一种调控基因表达的元件，包括人工串联启动子和其下游的RBS，所述人工串联启动子是将启动子P rpoB、P spoVG和P sigW中的至少两个进行串联，所述启动子P rpoB、P spoVG和P sigW的核苷酸序列分别如SEQ ID NO.1、SEQ ID NO.7和SEQ ID NO.11所示。
在本发明的一种实施方式中，所述人工串联启动子的核苷酸序列如SEQ ID NO.17-SEQ ID NO.27任一所示
在本发明的一种实施方式中，所述人工串联启动子的核苷酸序列如SEQ ID NO.29-SEQ ID NO.59任一所示。
在本发明的一种实施方式中，当在P rpoB和P spoVG启动子的核心区之间分别了插入了60bp、75bp的间隔序列，分别获得如SEQ ID NO.32、SEQ ID NO.33所示的新启动子P AW-D60、P AH-D75。
在本发明的一种实施方式中，当在P sigW、P rpoB和P spoVG的前两个启动子的核心区之间分别插入了45bp、75bp的间隔序列，分别获得如SEQ ID NO.43、SEQ ID NO.45所示的新启动子P WAH-D45、P WAH-D75。
在本发明的一种实施方式中，当在P rpoB、P sigW和P spoVG的前两个启动子的核心区之间分别插入了30bp、90bp的间隔序列，分别获得如SEQ ID NO.54、SEQ ID NO.58所示的新启动子P AWH-D30、P WAH-D90。
在本发明的一种实施方式中，所述RBS的核苷酸序列如SEQ ID NO.60-72任一所示。
在本发明的一种实施方式，目的基因的表达宿主包括枯草芽孢杆菌。
在本发明的一种实施方式，目的基因的表达宿主包括Bacillus subtilis 168、Bacillus subtilis WB400、Bacillus subtilis WB600或Bacillus subtilis WB800。
在本发明的一种实施方式，目的基因包括外源基因或内源基因。
在本发明的一种实施方式，目的基因包括酶基因或非酶基因。
本发明的第二个目的是提供含有上述元件的载体。
本发明的第三个目的是提供表达上述载体的基因工程菌。
本发明的第四个目的是提供一种调控枯草芽孢杆菌中目的基因表达的方法，将上述的调控元件与目的 基因共表达。
在本发明的一种实施方式中，所述目的蛋白包括酶。
在本发明的一种实施方式中，所述枯草芽孢杆菌包括Bacillus subtilis 168、Bacillus subtilis WB400、Bacillus subtilis WB600或Bacillus subtilis WB800。
本发明的第五个目的是提供上述的调控元件或基因工程菌在制备目的蛋白中的应用。
本发明的第六个目的是提供上述的调控元件或基因工程菌在食品、制药或化工领域的应用。
本发明的有益效果：本发明首先筛选和表征了不同sigma亚基识别启动子，从中选择被sigA、sigH和sigW亚基识别的活性最高(将含不同调控元件的重组质粒转化至枯草芽孢杆菌中，以重组菌培养后培养液的荧光强度表征目的基因表达量和调控元件的活性)的启动子，通过核心区的双串联和三串联、核心区间隔序列优化、RBS重设计等方式，获得了活性进一步提高的调控元件。
P AW-D60、P AH-D75荧光强度比改造前的P AW(荧光强度为20262a.u/OD 600)分别提高了0.94倍、1.03倍；P WAH-D45、P WAH-D75荧光强度(18245a.u./OD 600)比改造前的P WAH(荧光强度为10261a.u/OD600)分别提高了0.87倍、0.96倍；P AWH-D30、P WAH-D90荧光强度比改造前的P AWH(荧光强度为16879a.u/OD600)分别提高了0.78倍、0.78倍。
将P AH-D75与RBS11(SEQ ID NO.70)组合时，荧光强度可达76216a.u/OD 600，比P AH-D75提高了0.85倍；将P WAH-D75与RBS13(SEQ ID NO.72)组合时，荧光强度可达77751a.u/OD 600，比P WAH-D75提高了1.17倍；将P AWH-D30与RBS13(SEQ ID NO.72)组合时，荧光强度可达73781a.u/OD 600，比P AWH-D30提高了1.45倍.。
通过本发明提供的方法，我们可以用简便的启动子设计和改造方法获得活性更高、设计性和兼容性更强的启动子。该方法简单、易于实现，在构建外源蛋白高效表达系统和合成生物学研究中具有广阔的应用前景。
附图说明
图1：不同sigma亚基识别的天然内源启动子核心区筛选和表征。
图2：串联启动子构建和活性表征。
图3：串联启动子核心区间隔序列优化a：间隔序列优化示意图；b：P AH间隔序列优化；c：P WAH间隔序列优化；d：P AWH间隔序列优化。
图4：启动子与RBS兼容性检测，a：P rpoB启动子RBS设计；b：P spoVG启动子RBS设计；c：P sigW启动子RBS设计；d：P AH-D75启动子RBS设计；e：P WAH-D75启动子RBS设计；f：P AWH-D30启动子RBS设计。
具体实施方式
1、启动子克隆方法：设计包含启动子序列的引物，以带有sfGFP报告基因(Genbank ID：AVR55189.1)的大肠杆菌-枯草芽孢杆菌穿梭载体pB-sfGFP(即pBSG03，构建方法见Guan C,Cui W,Cheng J,et al.Construction and development ofan auto-regulatory gene expression system in Bacillus subtilis[J].Microbial Cell Factories,2015,14(1):150)为模板，用PrimeSTAR MAX DNA聚合酶(购自Takara，货号：R045Q)进行全质粒PCR，PCR程序为：预变性98℃1min，循环为变性98℃30s，退火50℃30s，延伸72℃1min，共30个循环，最后72℃延伸10min。之后用限制性内切酶DpnI消化去除质粒模板，将PCR产物纯化。之后用Infusion重组方法将片段环化，转化大肠杆菌JM109感受态细胞。
2、sfGFP荧光强度的检测方法：样品12000×g离心2min，收集菌体，PBS缓冲液洗3次，用PBS稀释到一定浓度的菌体悬液，取200μL至96孔酶标板，放入SynergyTM H4荧光酶标仪检测荧光。激发光485nm，吸收光528nm，检测荧光。
3、培养基：LB培养基(g.L -1)：胰蛋白胨10，NaCl 10，酵母提取物5，pH 7.0，配制固体培养基时添加琼脂粉20。
4、枯草芽孢杆菌168转化方法：挑单菌落枯草芽孢杆菌168接种至2mL的SPI培养基中，37℃摇床培养12-14h；从培养物中取100μL，接种至5mL SPI培养基中，37℃摇床培养4-5h后开始测OD 600。当OD 600约为1.0时，移取200μL菌液转接至2mL的SPII培养基中，于37℃、100r·min -1摇床孵育1.5h；向管中加入20μL l00×EGTA溶液，于37℃、100r·min -1摇床中培养10min后，每l.5mL离心管分装500μL；向管中加入经过测序验证正确的适量质粒，吹吸混匀放置于37℃、100r·min -1的摇床中培养2h；培养结束，吸取菌液约200μL均匀涂相应的选择性平板，37℃培养12-14h。
实施例1：不同sigma亚基识别单启动子的克隆和表征
选择P rpoB、P sucA、P mtnK、P ylbP、P ylxM和P yydE六个SigA亚基识别的启动子(核苷酸序列分别如SEQ ID NO.1-SEQ ID NO.6所示)，P spoVG、P pspoVS、P spo0M和P minC四个SigH亚基识别的启动子(核苷酸序列分别如SEQ ID NO.7-SEQ ID NO.10所示)和P sigW、P ydbS、P yobJ、P yqeZ、P ythP和P yuaF六个SigW识别的启动子(核苷酸序列分别如SEQ ID NO.11-SEQ ID NO.16所示)进行试验。克隆的启动子核心区包括以上启动子的-10区、-35区和转录起始位点(TSS)共70bp。待筛选和鉴定的启动子序列被设计在引物上(见表2)，通过全质粒PCR的方法将启动子序列引入载体骨架pB-sfGFP，之后通过DpnI消化、纯化和组装、转化构建出克隆的带有含单启动子sfGFP表达质粒。将表达单启动子的质粒转化至枯草芽孢杆菌168菌株中，获得的重组枯草芽孢杆菌在LB培养基中，37℃，200rpm下培养24h以检测sfGFP的表达水平，通过sfGFP的荧光信号高低判断启动子活性的高低。结果如图1和表3所示，在SigA识别的启动子中，P rpoB启动子活性最高，SigH识别的启动子中，P spoVG活性最高；SigW识别的启动子中，P sigW活性最高(图1)。
表2 单启动子克隆引物
引物 序列(5’-3’) a
P rpoB-1 CGGTATTTTAACTATGTTAATATTGTAAAATGCCAATGTATTCGAACATCATATTTAAAGTACGAGGAG
P rpoB-2 ACAATATTAACATAGTTAAAATACCGAGTCAAACTTTTTTTGCTTACCTGCCCTCTGCCACC
P sucA-1 ACAATCAAGGTAGAATCAAATTGCAAACAGTGGTAAAATATTCGAACATCATATTTAAAGTACGAGGAG
P sucA-2 TTGCAATTTGATTCTACCTTGATTGTTCACAAAATAGTAAAAAACACCTGCCCTCTGCCACC
P ylbP-1 TTTTTTAAATAAAGCGTTTACAATATATGTAGAAACAACAATCGAACATCATATTTAAAGTACGAGGAG
P ylbP-2 ATATTGTAAACGCTTTATTTAAAAAATCCAAATATTTAAACTTTAACCTGCCCTCTGCCACC
P ylxM-1 GTGTCATTAAAACCGTGTAAACTAAGTTATCGTAAAGGGATTCGAACATCATATTTAAAGTACGAGGAG
P ylxM-2 CTTAGTTTACACGGTTTTAATGACACTGTCAAGTTTTTATCTTGTACCTGCCCTCTGCCACC
P yydE-1 AAAGCAGTTATGCGGTACTATCATATAAAGGTCCAATGTTTTCGAACATCATATTTAAAGTACGAGGAG
P yydE-2 ATATGATAGTACCGCATAACTGCTTTTAGAGACAATTAAAACGAGACCTGCCCTCTGCCACC
P mtnK-1 CTAACTAAATTACCTGTTACCATGTTCATCAACTGATAAATTCGAACATCATATTTAAAGTACGAGGAG
P mtnK-2 AACATGGTAACAGGTAATTTAGTTAGTTGTCAATATATTTTTTAAACCTGCCCTCTGCCACC
P minC-1 GATTTTATCTTTTTTTGACGAAATGAGTATGTTGTTGAGGTTCGAACATCATATTTAAAGTACGAGGAG
P minC-2 TCATTTCGTCAAAAAAAGATAAAATCCTTTTTACTCATCTCTCAAACCTGCCCTCTGCCACC
P spoVG-1 TTTCAGAAAAAATCGTGGAATTGATACACTAATGCTTTTATTCGAACATCATATTTAAAGTACGAGGAG
P spoVG-2 TATCAATTCCACGATTTTTTCTGAAATCCTGCTCGTTTTTAAAATACCTGCCCTCTGCCACC
P spoVS-1 GAATATAGCAACTCCTTAGTGAATATAGTAAAAATGGAAGGTCGAACATCATATTTAAAGTACGAGGAG
P spoVS-2 ATATTCACTAAGGAGTTGCTATATTCCTGCTTTTCTTTTTAATATACCTGCCCTCTGCCACC
P spo0M-1 GAAAAAAGTATGAATCAAACGAATCTTTTTTCCTCCTTCTTTCGAACATCATATTTAAAGTACGAGGAG
P spo0M-2 AGATTCGTTTGATTCATACTTTTTTCCTATTATTCGTCTCGGCCTACCTGCCCTCTGCCACC
P sigW-1 ACCTTTTGAAACGAAGCTCGTATACATACAGACCGGTGAAGTCGAACATCATATTTAAAGTACGAGGAG
P sigW-2 TGTATACGAGCTTCGTTTCAAAAGGTTTCAATTTTTTTATAAAATACCTGCCCTCTGCCACC
P ydbS-1 ACCTTTCTGTAAAAGAGACGTATAAATAACGACGAAAAAAATCGAACATCATATTTAAAGTACGAGGAG
P ydbS-2 TTTATACGTCTCTTTTACAGAAAGGTTTCATTCTTAAGCATACAGACCTGCCCTCTGCCACC
P yobJ-1 ACCTTTTTTATTTTAGCCCGTATTAAAAGTAAATTCAGAGATCGAACATCATATTTAAAGTACGAGGAG
P yobJ-2 TTAATACGGGCTAAAATAAAAAAGGTTTCATATAAAACGGGACTAACCTGCCCTCTGCCACC
P yqeZ-1 AACCTTTGATACATTTGTTACGTATGAAGAGAAGGCACTTATCGAACATCATATTTAAAGTACGAGGAG
P yqeZ-2 CATACGTAACAAATGTATCAAAGGTTTCATTTTTTTATGTATAAAACCTGCCCTCTGCCACC
P ythP-1 AAACTTTTTTTATTCTATTTCGTAGTAAATTTTGGAGGTGATCGAACATCATATTTAAAGTACGAGGAG
P ythP-2 ACTACGAAATAGAATAAAAAAAGTTTCTTTAACCATAATAATATTACCTGCCCTCTGCCACC
P yuaF-1 ACTTTTCCCGAGGTGTCTCGTATAAATGGTAACGGCAGCCGTCGAACATCATATTTAAAGTACGAGGAG
P yuaF-2 TTTATACGAGACACCTCGGGAAAAGTTTCAAAATTTTAAGACAAAACCTGCCCTCTGCCACC
表3 带有含单启动子sfGFP表达质粒的重组菌培养24h后的总荧光强度
Figure PCTCN2019078816-appb-000001
实施例2：串联启动子的构建和表征
将P rpoB、P spoVG和P sigW进行串联设计，用表5中的引物以实施例1中构建的带有启动子P rpoB、P spoVG和P sigW的三种质粒作为模板做全质粒PCR构建含串联启动子并表达sfGFP的质粒，将串联启动子按照串联核心区的类型和顺序命名，得到双串联启动子P AH、P AW、P HA、P HW、P AW、P WA和P WH(核苷酸序列分 别如SEQ ID NO.17-SEQ ID NO.22所示)，三串联启动子P AHW、P AWH、P HAW、P HWA、P WAH和P WHA(核苷酸序列分别如SEQ ID NO.23-SEQ ID NO.28所示)，构建引物见表5。将构建好的重组质粒转化枯草芽孢杆菌168，获得的重组枯草芽孢杆菌在LB培养基中，37℃，200rpm下培养，6h、12h、24h后检测荧光信号，通过sfGFP的荧光信号高低判断启动子活性的高低。
表5 串联启动子构建引物
Figure PCTCN2019078816-appb-000002
表6 带有含双串联和三串联启动子sfGFP表达质粒的重组菌培养后的总荧光强度
Figure PCTCN2019078816-appb-000003
带有含双串联和三串联启动子sfGFP表达质粒的重组菌培养6、12、24h后的荧光强度见图2和表6，双串联启动子和三串联启动子的活性较单启动子大部分都体现出了不同程度的提高，并且三串联启动子大部分活性高于双串联启动子，其中P WHA启动子质粒转化枯草芽孢杆菌不成功。选择双串联启动子中活性较高的P AH、三串联启动子中活性较高的的P WAH和P AWH作为下一步改造材料。
实施例3：串联启动子间隔序列优化
在启动子核心区之间插入不同长度的间隔序列(图3a)，将间隔序列长度设定为15bp、30bp、45b、60bp、75bp和90bp，得到AH-D15、AH-D30、AH-D45、AH-D60、AH-D75、AH-D90、WAH-U15、WAH-U30、WAH-U45、WAH-U60、WAH-U75、WAH-U90、WAH-D15、WAH-D30、WAH-D45、WAH-D60、WAH-D75、WAH-D90、AWH-U15、AWH-U30、AWH-U45、AWH-U60、AWH-U75、AWH-U90、AWH-D15、AWH-D30、AWH-D45、AWH-D60、AWH-D75、AWH-D90、AWH-DU30(核苷酸分别序列如SEQ ID NO.29-SEQ ID NO.59所示)。将SEQ ID NO.29-SEQ ID NO.59所示的启动子序列分别克隆在pB-sfGFP载体上，之后将重组质粒转化枯草芽孢杆菌168进行检测，获得的重组枯草芽孢杆菌在LB培养基中，37℃，200rpm下培养24h后检测荧光信号，通过sfGFP的荧光信号高低判断启动子活性的高低。
结果显示，如P AW-D60、P AH-D75荧光强度比改造前的P AW(荧光强度为20262a.u/OD 600)分别提高了0.94倍、1.03倍；P WAH-D45、P WAH-D75荧光强度(18245a.u./OD 600)比改造前的P WAH(荧光强度为10261a.u/OD600)分别提高了0.87倍、0.96倍；P AWH-D30、P WAH-D90荧光强度比改造前的P AWH(荧光强度为16879a.u/OD600)分别提高了0.78倍、0.78倍。
(图3b、3c和3d；表8)。结果说明，在串联的核心区之间插入合适长度的间隔序列可以有效进一步提升串联启动子的活性。
表8 含插入间隔序列的双串联和三串联启动子的重组质粒的重组菌培养24h后的荧光强度
启动子 荧光强度(a.u./OD 600)
P AH 20262
P AH-D15 32865
P AH-D30 33033
P AH-D45 34869
P AH-D60 39476
P AH-D75 41154
P AH-D90 28592
P WAH 18245
P WAH-U15 18278
P WAH-U30 18553
P WAH-U45 17198
P WAH-U60 18161
P WAH-U75 19295
P WAH-U90 18419
P WAH-D15 24711
P WAH-D30 25307
P WAH-D45 34100
P WAH-D60 32029
P WAH-D75 35819
P WAH-D90 24355
P AWH 16879
P AWH-U15 16707
P AWH-U30 15762
P AWH-U45 16157
P AWH-U60 15852
P AWH-U75 16770
P AWH-U90 18045
P AWH-D15 27627
P AWH-D30 30084
P AWH-D45 28409
P AWH-D60 25195
P AWH-D75 26580
P AWH-D90 29999
P AWH-DU30 24562
实施例4：启动子和RBS兼容性研究
将实施例1和实施例3中的启动子P rpoB、 PspoVG、P sigW、P AH-D75、P WAH-D75和P AWH-D30与13个RBS分别进行组合。将RBS1-13序列(核苷酸序列分别如SEQ ID NO.60-SEQ ID NO.72所示)分别克隆在含有串联启动子的载体上，之后将重组质粒转化至枯草芽孢杆菌168，获得的重组枯草芽孢杆菌在LB培养基中，37℃，200rpm下培养24h后检测荧光信号，通过sfGFP的荧光信号高低判断调控元件活性的高低。之后对不同组合的RBS理论强度和实际测定的荧光值进行相关性分析，以r值评价相关性。结果如图4所示，单启动子P ropB和RBS组合设计的相关性不高，而串联启动子和RBS组合之后的相关性均高于单启动子P ropB和RBS的组合。说明串联启动子设计RBS可以提高启动子和RBS元件组合使用的兼容性，即增强了表达外源蛋白时的可设计性和可预测性。
如表10所示，将P AH-D75与RBS11(SEQ ID NO.70)组合时，荧光强度可达76216a.u/OD 600，比P AH-D75提高了0.85倍；将P WAH-D75与RBS13(SEQ ID NO.72)组合时，荧光强度可达77751a.u/OD 600，比P WAH-D75提高了1.17倍；将P AWH-D30与RBS13(SEQ ID NO.72)组合时，荧光强度可达73781a.u/OD 600，比P AWH-D30提高了1.45倍.。说明串联启动子设计RBS还可以增强目的基因的表达。
表10 启动子分别组合RBS1-13后的翻译起始效率和荧光强度
Figure PCTCN2019078816-appb-000004
Figure PCTCN2019078816-appb-000005
Figure PCTCN2019078816-appb-000006
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2019078816-appb-000007
Figure PCTCN2019078816-appb-000008
Figure PCTCN2019078816-appb-000009
Figure PCTCN2019078816-appb-000010
Figure PCTCN2019078816-appb-000011
Figure PCTCN2019078816-appb-000012
Figure PCTCN2019078816-appb-000013
Figure PCTCN2019078816-appb-000014
Figure PCTCN2019078816-appb-000015
Figure PCTCN2019078816-appb-000016
Figure PCTCN2019078816-appb-000017
Figure PCTCN2019078816-appb-000018
Figure PCTCN2019078816-appb-000019
Figure PCTCN2019078816-appb-000020
Figure PCTCN2019078816-appb-000021
Figure PCTCN2019078816-appb-000022
Figure PCTCN2019078816-appb-000023
Figure PCTCN2019078816-appb-000024
Figure PCTCN2019078816-appb-000025
Figure PCTCN2019078816-appb-000026
Figure PCTCN2019078816-appb-000027

Claims (14)

  1. 一种调控目的基因表达的方法，其特征在于，将调控元件与目的基因共表达，所述调控元件包括人工串联启动子和其下游的RBS，所述人工串联启动子是将启动子P rpoB、P spoVG和P sigW中的至少两个进行串联，所述启动子P rpoB、P spoVG和P sigW的核苷酸序列分别如SEQ ID NO.1、SEQ ID NO.7和SEQ ID NO.11所示。
  2. 如权利要求1所述的方法，其特征在于，所述人工串联启动子的核苷酸序列如SEQ ID NO.17-SEQ ID NO.27任一所示。
  3. 如权利要求1所述的方法，其特征在于，所述人工串联启动子的核苷酸序列如SEQ ID NO.29-SEQ ID NO.59任一所示。
  4. 如权利要求1所述的方法，其特征在于，所述RBS的核苷酸序列如SEQ ID NO.60-72任一所示。
  5. 如权利要求1所述的方法，其特征在于，目的基因的表达宿主包括枯草芽孢杆菌。
  6. 如权利要求5所述的方法，其特征在于，目的基因的表达宿主包括Bacillus subtilis 168、Bacillus subtilis WB400、Bacillus subtilis WB600或Bacillus subtilis WB800。
  7. 如权利要求1所述的方法，其特征在于，目的基因包括外源基因。
  8. 如权利要求1所述的方法，其特征在于，目的基因包括内源基因。
  9. 如权利要求1所述的方法，其特征在于，目的基因包括酶基因或非酶基因。
  10. 如权利要求1所述的方法，其特征在于，应用于食品领域。
  11. 如权利要求1所述的方法，其特征在于，应用于保健品领域。
  12. 如权利要求1所述的方法，其特征在于，应用于制药领域。
  13. 一种调控基因表达的元件，其特征在于，包括人工串联启动子和其下游的RBS，所述人工串联启动子是将启动子P rpoB、P spoVG和P sigW中的至少两个进行串联，所述启动子P rpoB、P spoVG和P sigW的核苷酸序列分别如SEQ ID NO.1、SEQ ID NO.7和SEQ ID NO.11所示。
  14. 表达权利要求13所述元件的基因工程菌。
PCT/CN2019/078816 2018-12-17 2019-03-20 一种构建高效枯草芽孢杆菌启动子的方法 Ceased WO2020124831A1 (zh)

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